Metabolic Brain Disease

, Volume 30, Issue 5, pp 1193–1206 | Cite as

Possible cause for altered spatial cognition of prepubescent rats exposed to chronic radiofrequency electromagnetic radiation

  • Sareesh Naduvil Narayanan
  • Raju Suresh Kumar
  • Kalesh M. Karun
  • Satheesha B. Nayak
  • P. Gopalakrishna Bhat
Research Article

Abstract

The effects of chronic and repeated radiofrequency electromagnetic radiation (RFEMR) exposure on spatial cognition and hippocampal architecture were investigated in prepubescent rats. Four weeks old male Wistar rats were exposed to RF-EMR (900 MHz; SAR-1.15 W/kg with peak power density of 146.60 μW/cm2) for 1 h/day, for 28 days. Followed by this, spatial cognition was evaluated by Morris water maze test. To evaluate the hippocampal morphology; H&E staining, cresyl violet staining, and Golgi-Cox staining were performed on hippocampal sections. CA3 pyramidal neuron morphology and surviving neuron count (in CA3 region) were studied using H&E and cresyl violet stained sections. Dendritic arborization pattern of CA3 pyramidal neuron was investigated by concentric circle method. Progressive learning abilities were found to be decreased in RF-EMR exposed rats. Memory retention test performed 24 h after the last training revealed minor spatial memory deficit in RF-EMR exposed group. However, RF-EMR exposed rats exhibited poor spatial memory retention when tested 48 h after the final trial. Hirano bodies and Granulovacuolar bodies were absent in the CA3 pyramidal neurons of different groups studied. Nevertheless, RF-EMR exposure affected the viable cell count in dorsal hippocampal CA3 region. RF-EMR exposure influenced dendritic arborization pattern of both apical and basal dendritic trees in RF-EMR exposed rats. Structural changes found in the hippocampus of RF-EMR exposed rats could be one of the possible reasons for altered cognition.

Keywords

Mobile phone Cognition Prepubescent rat Hippocampus Morris water maze 

Introduction

The primary vision for the mobile and personal telecommunication services and systems is to enable communication with a person at any time at any place and in any form. While implementing this, it is mandatory to screen the possible health effects (if any) due to these technologies. The effects of radiofrequency electromagnetic radiation (RF-EMR) on biological systems depend both on the power and frequency of radiation (IARC 2011). Biological effects of RF-EMR have been studied in various model organisms by several researchers (Finnie et al. 2002; Aksoy et al. 2005; Chavdoula et al. 2010; Partsvania et al. 2013). Nevertheless, until today studies have not been able to categorically prove whether RF-EMR exposure is harmful to humans or not. On the other hand, its possible health effects couldn’t be ruled out. At present, the regulatory bodies adopt a precautionary policy while dealing with RF-EMR (WHO 2011).

Whether RF-EMR affects cognition in animals and also in humans is a puzzle to be solved. Cognition is the mental action of acquiring knowledge and understanding through thought, experience and senses. In rodents, spatial cognitive processing allows the formation of cognitive maps of their environment and this speed up the process of learning and memory in them. Spatial memory has been represented as working (short-term) and reference (long-term) memory (Good. 2002). Short-term memory (working memory) allows one to temporarily store and processes the information or task and that it retains for a short duration. Following this the particulars of the spatial information is discarded as this information is no longer required. Contrary to this, long term memory (reference memory) is retained longer as it is inevitable to complete subsequent tasks (Olton and Papas. 1979). Many reports are available addressing the influence of RF-EMR on cognition but inconsistent results do exist in these reports. Kumlin et al. (2007) have reported that, 900 MHz exposure (2 h/day, 5 days/week for 5 weeks), did enhance spatial memory performance and it did not affect hippocampal morphology in rats. On the other hand Fragopoulou et al. (2010) have found that, 2 h daily dose of 900 MHz radiation (pulsed GSM) from a mobile phone for 4 days induced altered spatial memory performance in mice. Another report suggest that, a single 15 min 900 MHz radiation (GSM; SAR = 6 W/kg) exposure did not induce astrocyte activation but increased IL-1β in the olfactory bulb and enhanced contextual emotional memory in GSM-exposed middle-aged rats (Bouji et al. 2012). In contrast to this another report suggest that, 916 MHz, 10 w/m2 EMF could influence learning and memory in rats to some extent in a period during exposure, and the rats might get adapted to long-term exposures (Hao et al. 2013). RF-EMR exposure (phone was kept in vibratory mode while exposing to rats) for a period of 4 weeks induced deficits in spatial memory performance (Narayanan et al. 2009). Jiang et al. (2013) have reported that, RF-EMR exposure induced, cognitive and memory impairment, oxidative stress and overexpression of beta amyloid protein in rat hippocampus. Furthermore, a very recent study by Saikhedkar et al. (2014) demonstrated that, 900 MHz exposure (4 h/day) for a period of 15 days induced learning and memory deficits and this was correlated with hippocampal neuronal degeneration in rats.

Effect of RF-EMR exposure on human cognition is also complicated with inconsistent results across reports. Khorseva et al. (2011) have reported that, an increased number of phonemic perception disorders, abatement of efficiency, reduced indicators for the arbitrary and semantic memory, and an increased fatigue was found with children (7–12 years old) using mobile communication compared to control. Loughran et al. (2013) could not find a clear significant effects neither on the waking EEG nor on cognitive performances of adolescents and this indicate that, adolescent do not appear to be more sensitive compare to adults to RF-EMR. Additionally, 900 MHz radiation (GSM) did not affect the performance in the selective attention test and the n-back task in young males (Sauter et al. 2011). A systematic review and meta-analysis affirms that, RF-EMR do not seem to affect cognitive or psychomotor performance but it warrant a need for extensive study on neglected issues such as investigation of repeated, intensive and chronic exposures in sensitive populations such as children (Valentini et al. 2010).

The effects of RF-EMR exposure on cognitive performance in early adolescent or prepubescent rats is still a subject to debate. In rodents adolescence has three stages; prepubescent/early adolescent (day 21–34), mid adolescent (day 34–46), late adolescent (day 46–59). Report indicates that, significant brain development (particularly frontal lobe) occurs during adolescence (O’Donnell, et al. 2005). Studies revealed that, habituation of stress response is seen with adult rats when exposed to chronic stress repeatedly, but prolonged activation is seen with juvenile rats exposed to the same stressors (Vazquez and Akil. 1993). Furthermore, HPA axis responses to acute and chronic stress depend on the developmental stage of the rats (Avital and Richter-Levin. 2005). The adolescent brain especially hippocampus might be more susceptible to stressors, other toxic agents, and high levels of glucocorticoids. The possible reason for this argument is that, though the adolescent brain undergoes active maturation, in rats the hippocampus continues to grow until adulthood (Lupien et al. 2009). Earlier we have reported that, RF-EMR exposure (1 h/day; phone was kept in vibratory mode) for a period of 4 weeks can induce a change in spatial memory performance in rats (Narayanan et al. 2009). Although a change in spatial cognition of rats was noted earlier, it is mandatory to evaluate whether these effects are attributed to RF-EMR alone as the phone was kept in vibratory mode while exposing to rats in the above mentioned study. The current study was designed to rule out this confounding factor and also to investigate other possible effects of RF-EMR on hippocampus. Specifically, in the current study, the influence of chronic and repeated RF-EMR exposure on cognitive performances and morphology of dorsal hippocampal CA3 pyramidal neurons were investigated in prepubescent rats.

Materials and methods

Animals and maintenance

Four weeks old male Wistar rats (weighing 50–60 g) were obtained from institutional Central Animal Research Facility (CARF). They were housed in polypropylene cages measuring 41 × 28 × 14 cm. Each of these cages had three rats housed. Rats were fed with water and food ad libitum and maintained in 12:12 h L:D environment, in an air-conditioned room. Institutional Animal Ethics Committee (IAEC) has approved all procedures used in the study.

Experimental groups and study design

Animals used for each experiment were obtained from six different litters. They were randomly assigned into the following groups. Control: Animals of this group remained in the home cage throughout the experimental period (28 days). Sham exposed: Animals of this group was exposed to mobile phone in switch off mode (1 h/day) for 28 days. RF-EMR exposed: They were exposed to RF-EMR (900 MHz) from an activated Global System for Mobile communications (GSM) mobile phone for 1 h/day (in silent mode; “no ring tone”) for 28 days. The entire study was carried out in two different stages; stage- I and II. Stage-I (analysis of spatial learning and memory); in this stage a total of 36 rats were used and were assigned into various groups (n = 12 in each group) as mentioned above. After the experimental period spatial cognition was tested (between 4.00 p.m. and 8.00 p.m.) using Morris water maze test. Stage-II (analysis of cellular changes in the hippocampal CA3 sub-region); thirty six male Wistar rats were used for this stage and they were assigned into different groups (n = 12 in each group) as mentioned above. On day 29, six animals from each group were euthanized to study hippocampal morphology (H&E staining and cresyl violet staining). Remaining animals were euthanized to study dendritic arborization using Golgi-Cox staining.

Whole body RF-EMR exposure and power dosimetry

The exposure mode and dose were replicated in this study with respect to earlier published reports (Narayanan et al. 2010, 2013) with minor modifications. Briefly, animals of RF-EMR group (3 in each home cage) were exposed to RF-EMR from an activated mobile phone (GSM; 900 MHz) with a permitted power level of 2 W and SAR specification 1.15 W/kg. While exposing mobile phone to rats it was inserted in a wire mesh cage (12 × 7 × 7 cm) and placed at the center of the animal home cage. The purpose of doing this was to prevent the effect of heat emitting from the phone on rats. The phone was continuously activated by giving unattended calls (50 calls/h) during the exposure period. Duration of exposure was 1 h/day (7 days in a week) for 28 days. This was done using a mobile phone auto dialer unit (indigenously made for the current study), which can dials four phones at a time. The contact between dialing phones and call receiving phones can be changed from one to four with respect to the need. All call receiving phones were purchased from the same manufacturer and with same SAR specification as mentioned earlier. The power density in the vicinity of the mobile phone was determined by using a spectrum analyzer (SPECTRAN HF-2025E with MCS Real-Time Spectrum Analyzer Software, Aaronia AG, Germany). When the phone was ringing, the peak power density recorded in its vicinity (3 cm away from mobile phone) was turned out to be 146.60 μW/cm2 (Narayanan et al. 2013).

Body weight measurements

Body weight was measured at 0 day (before the start of the experiment), 29th day (after the experimental period; before the start of MWM test) and 37th day (after MWM test), in various groups.

Analysis of spatial learning and memory

Spatial cognition testing was performed using Morris water maze test as described by Rudi and Peter (2001) with modifications. The apparatus is a water tank measuring 180 cm in diameter and 75 cm in depth. The tank was filled with water (22 ± 2 °C) to an approximate depth of 50 cm. By designating four points (north; N, south; S, east; E, and west; W) on the rim of the pool, it was divided into four quadrants. A platform (10 × 10 cm) was placed (submerged 2 cm below the water surface) in one of the quadrants and this quadrant was designated as “target quadrant” (TQ). To make the water opaque, just before the start of the experiment a nontoxic white paint powder was added. A black and white picture was hung on the wall and this allowed the rats to develop a spatial map strategy. Throughout the Morris water maze test session, the position of this cue was kept unchanged.

Spatial acquisition stage (spatial learning stage)

In the MWM, all rats were trained in a total of 6 sessions on 6 consecutive days (one session per day). Each training session included four swimming trials starting from four different quadrants of the maze. During each trial, the rat was released into the water in such a way that it faced the wall of the MWM. The spot of release of each rat to the maze was varied between N, W, S, and then E. However, the target quadrant remained the same throughout the entire test. During MWM training trial each rat was trained for a period of 3 min to find out the platform. In each trial, the escape latency was recorded from which the mean daily (session) escape latency of one rat was calculated. After locating the platform each rat was allowed to be on it for 15 s. If a rat was unable to locate the submerged platform within 3 min time, the experimenter guided the rat towards the platform and permitted it to be there for 15 s. After every trial each rat was taken out from MWM and dried with a towel and placed back in its home cage for 5 min rest before the next trial. Before the memory retention tests, every rat had a total of 24 trials.

Memory retention test-1 (without hidden platform)

This test was conducted 24 h after the last training session and it was lasted only 30 s. As mentioned above, before the start of the test, the maze water was made opaque and the submerged platform was also removed. In this test each rat was released in to the maze from one of the quadrant as described in the acquisition phase and its behaviour in the maze was video recorded and analyzed. The real time video records were obtained by a video camera (Sony) which was linked to a PC installed with Panlab SMART, (version 2.5) Image analysis system, Barcelona, Spain, for measuring various parameters. Parameters such as escape latency to reach target quadrant, distance swam in the target quadrant, number of entries and time spent in were studied.

Memory retention test-2 (with hidden platform)

This was conducted 48 h after the last trail. Duration of the test remained 30 s as in retention test-1. During this test the hidden platform was again placed in the TQ as in learning trial (Setlow and McGaugh. 2000). Each animal was released into the opposite quadrant of the TQ and escape latency to reach target quadrant, time spent in target quadrant and distance swam before reaching the platform (swim path distance) were recorded.

Hematoxylin & Eosin (H&E) staining (to analyze the gross cellular architecture)

Rats were euthanized by cervical dislocation. The brain was carefully dissected out, fixed in 10 % formalin and processed for paraffin embedding. Sections were cut at 6 μm thickness using a rotary microtome (Leica RM2155, Germany). Paraffin embedded sections were stained with H&E as per the standard procedure (Bancroft and Stevens 1990).

Analysis of dorsal hippocampal CA3 neurons

Six H&E stained sections of dorsal hippocampal regions were used for qualitative analysis. This was carried out in CA3 neurons of the dorsal hippocampus of left hemisphere. The hippocampal CA3 pyramidal cells were thoroughly examined for Hirano’s bodies and granulovacuolar bodies. This was done under 400× magnification using a light microscope (Magnus MLX, Microscope) by a trained individual blinded to experimental conditions. Though the significance of Hirano’s bodies (HB) and granulovacuolar bodies (GVB) is unknown, they are seen in Alzheimer’s disease.

Cresyl violet (CV) staining (to analyze the degree of neuronal survival)

Cresyl violet stain was prepared by dissolving 100 mg of cresyl violet (CV) powder (obtained from Sigma and Aldrich, USA) in warm (60 °C) distilled water. Once the CV powder was completely dissolved, 8–10 drops of glacial acetic acid was added to the solution and stirred well. This final solution was filtered using a filter paper and was stored at room temperature for further use. Paraffin embedded sections (prepared as described under H & E staining) were stained with cresyl violet as per the standard procedure (Bancroft and Stevens. 1990). Briefly, the sections were immersed in xylene for 5 min to remove wax (two changes). Followed by this, sections were immersed in 100 % alcohol for a period of 5 min. They were then washed in distilled water for 10–15 min. Following this, sections were immersed in CV solution for 10–15 min. The temperature of the CV stain solution was maintained 58 °C throughout the staining process. After this the sections were dehydrated through alcohol series (1 dip each in 90 & 100 % alcohol). Following this the sections were cleared using xylene for 2–3 min and later mounted under a cover slip using DPX.

Quantification of surviving neurons at CA3 region

Quantitative analysis of the CV stained slides were done after an independent person coding the slides. The light microscope (Magnus MLX, Microscope) was calibrated by an ocular micrometer and objective (stage) micrometer (Erma, Tokyo, Japan) before quantifying the surviving neurons in the CA3 region. 100 divisions in ocular micrometer turned out to be 250 μm on the stage. Total number of neurons in a field of 250 μm2 was counted. After placing the ocular micrometer in the microscope eye piece, the number of normal neuron cell bodies (with normal cell membrane, nucleus and not darkly stained) present between 0 and 100 divisions of ocular micrometer were counted manually from ten brain sections of each animal. Each group had six animals and therefore a total of 60 brain sections were analyzed from each group. The quantitative analysis was carried out in CA3 regions of the dorsal hippocampus of left hemisphere. The slides were decoded only after the completion of statistical comparison of various test groups values.

Golgi-Cox staining (to analyze the dendritic arborization)

All reagents used for this staining were obtained from Sigma and Aldrich, USA. After the experimental period cervical dislocation was performed to euthanize the rats. The whole brain was dissected out and impregnated in Golgi-Cox solution for 2 weeks and processed for Golgi-Cox staining as reported earlier (Narayanan et al. 2014b).

Dendritic quantification

Hippocampal CA3 neurons (10 per animal, 60 in a group) were traced under 400× magnification using a Camera Lucida (LABKRON, India) fixed to a monocular microscope.

Criteria for the selection of neurons for Camera Lucida tracing and analysis

Neurons from hippocampus CA3 region was selected for Camera Lucida tracing is based on the following criteria.
  1. a.

    Neurons must be confined to the CA3 regions of the hippocampus.

     
  2. b.

    Neurons selected must be darkly stained and the entire dendritic profile should be identifiable.

     
  3. c.

    No truncation of any branch should be present within 100 μm radius from the soma.

     
  4. d.

    Neurons selected should be comparatively isolated from neighboring neurons.

     

Quantification of dendritic branching points and dendritic intersections

Apical and basal dendritic branching points and dendritic intersections of CA3 pyramidal neurons were quantified by concentric circles method (Sholl 1953). Both branching points and intersections were counted up to a distance of 100 μm from soma (Fig. 1).
Fig. 1

Camera Lucida tracing depicting a hippocampal CA3 pyramidal neuron and the scheme of dendritic quantification

Statistical analysis

The descriptive statistics of various outcome measures were represented as Mean ± SE. Significant difference between the groups in MWM retention test and neuron quantification were analyzed by ANOVA test. Followed by this, Tukey’s test was performed for multiple comparisons. To find out the intergroup difference in MWM learning and body weight gain, repeated measures of ANOVA followed by post-hoc Tukey’s test was performed. p < 0.05 was considered as statistically significant at 5 % level of significance. SPSS statistical package (version 16.0) was used for data analysis.

Results

Effect of RF-EMR on body weight gain in rats

All groups had significant weight gain on 29th day compared to 0-day and this was found to be statistically significant (Table 1). No group difference was observed in body weight of rats when measured on 29th day. However, the mean value was found to be slightly more in RF-EMR group on 29th day. MWM test had an impact on body weight as demonstrated by their loss of weight after MWM test. The weight loss (on 37th day) was not very much evident in control and sham but this was very evident in RF-EMR group and this difference was found to be statistically significant compared to 29th day weight in the same group but not with other groups.
Table 1

RF-EMR effects on body weight

Group

Body weight (g)

On day- 0

On day- 29

On day- 37

Control

55.83 ± 1.57

107.30 ± 3.71***

105.50 ± 2.48

Sham

58.58 ± 3.22

115.10 ± 3.32***

109.80 ± 3.32

RF-EMR exposed

57.00 ± 3.08

121.90 ± 2.53***

112.50 ± 2.91§§

***p < 0.001, §§ p < 0.01

Analysis of spatial learning and memory

Spatial learning (latency to reach the target)

MWM acquisition data from hidden platform task for a period of six consecutive days are represented in Fig. 2. It can be noticed that all group animals had learned the task well as demonstrated by their decreasing escape latency to reach the submerged platform from day 1 to 6. Though RF-EMR exposed group animals had learnt this task, their escape latencies were slightly towards higher side as compared to control and sham exposed rats on certain days. This difference was found to be more evident on day 4. On this day the mean escape latency was found to be ~30 s in RF-EMR exposed group but this was ~20 s in control and sham exposed groups. Repeated measures of ANOVA followed by post-hoc comparisons affirmed a notable difference between the means of RF-EMR exposed group compared to control and sham exposed. The mean escape latency of rats continued to be higher in RF-EMR rats on 5th and 6th day compared to control and sham exposed group. Repeated measures of ANOVA and multiple comparison tests revealed a significant difference between groups in the acquisition latencies on 5th and 6th day.
Fig. 2

Morris water maze escape latencies from day 1 to 6. Improved progressive learning is seen in all groups as indicated by their decreasing escape latencies from 1st to 6th day. However, deficit in progressive learning abilities were seen in RF-EMR exposed group from day 4–6 compared to others. (***p < 0.001, ∂∂∂p < 0.001)

Spatial memory retention test-1 (without hidden platform)

Latency to reach the TQ

RF-EMR exposed rats had an increased escape latency to reach the TQ compared to control and sham exposed rats (Fig. 3a). However, this difference was not statistically significant. This indicates that, they did learn the paradigm to some extent and had retention about the spatial orientation 24 h after the final trial session.
Fig. 3

Latency to reach the TQ (a), percentage of time spent in the TQ (b), distance swam in the TQ (c), entries to TQ (d) and representative animal trajectories (e 1–3) during MWM memory retention test-1 Note; B- begin, E- end, TQ- target quadrant

Percentage of time spent in the TQ

RF-EMR exposed rats spent slightly less time in the TQ compared to control and sham exposed rats but this was not statistically significant (Fig. 3b).

Distance swam in the TQ

The distance swam by control rats in the TQ was found to be 306.80 ± 33.65 cm in and 292.70 ± 17.22 cm in sham exposed rats. This was reduced in RF-EMR exposed rats to 227.90 ± 16.21 cm (Fig. 3c and e-3) but it was not found to be statistically significant. The real time videotaped records showed the path taken by animals during memory retention test-1 and it revealed the decreased distance travelled by RF-EMR rats in the TQ compared to control and sham exposed rats (Fig. 3e-3).

Entries to TQ

The number of entries to TQ was found to be less in RF-EMR exposed group compared to others but this difference was not statistically significant. These results indicate that, RF-EMR exposed rats showed a mild deficit in spatial memory retention when tested 24 h after the last trial (Fig. 3d).

Spatial memory retention test-2 (with hidden platform)

Latency to reach the TQ

As depicted in Fig. 4, the latency to reach TQ (when tested 48 h after the last training session) was not significantly affected in rats exposed to RF-EMR (4A). Statistical analysis revealed no significant difference between groups in the escape latency to reach the TQ (p = 0.094).
Fig. 4

Latency to reach the TQ (a), latency to reach the target (b), distance swam to reach the target (c) and representative animal trajectories (d 1–3) during MWM retention test-2. Note; B- begin, E- end, TQ- target quadrant. *p < 0.05, ∂p < 0.05

Latency to reach the target

RF-EMR exposed rats had a higher escape latency to reach the target compared to control and sham exposed rats. The control rats and sham exposed rats had taken 9.80 ± 2.37 and 9.70 ± 2.65 s respectively to reach the target during the retention test. This was considerably increased in RF-EMR exposed rats. It was found to be 20.37 ± 3.38 s in this group (Fig. 4b). Statistical tests affirmed a significant difference between the mean values of RF-EMR group compared to control and sham exposed group.

Distance swam to reach the target

Distance swam before finding the submerged platform was significantly increased in RF-EMR exposed rats compared to control and sham exposed group. The mean distance swam by the control rats was 270.50 ± 45.45 cm and that of sham exposed group was 276.90 ± 63.94 cm. It was significantly increased in RF-EMR exposed group and the value was found to be 495.50 ± 73.06 cm (Fig. 4c). Statistical tests revealed a significant difference between the groups. The real time videotaped records indicated this behaviour of rats during the retention test-2 (Fig. 4d-3). The retention test-2 results clearly demonstrate that, the spatial memory which had formed after several trials and primarily expressed during the performance of a task was affected due to RF-EMR exposure.

Analysis of dorsal hippocampal CA3 neurons

Analysis of the H&E stained hippocampal CA3 pyramidal neurons did not reveal the presence of Hirano’s bodies, and Granulovacuolar bodies in any of the groups studied.

Analysis of the degree of neuronal survival in CA3 region

Qualitative analysis of cresyl violet stained hippocampus was carried out prior to the quantification and this revealed the sparse appearance of hippocampal CA3 region in RF-EMR exposed group (Fig. 5c) compared to other groups (Fig. 5a and b). Examination of CA3 regions in high magnification revealed compactly arranged, healthy, pyramidal cells (with clear nucleus, intact cell membrane and a diameter of ~8–10 μm) in control and sham exposed rats (Fig. 5d and e), but RF-EMR exposed rat hippocampus failed to show such an arrangement (Fig. 5f). Also in the RF-EMR exposed group, outline of CA3 region (CA3 cell band) was fuzzy and along with the normal cells darkly stained, unhealthy neurons were also found (Fig. 5f). Quantification of healthy neurons in the CA3 region affirmed a notable difference between groups in the number of neurons. It was found to be reduced in RF-EMR exposed rats compared to other groups (Fig. 5g).
Fig. 5

Representative photomicrographs of cresyl violet stained hippocampal CA3 region from control (a and d), sham exposed (b and e) and RF-EMR exposed (c and f) rats. CA3 surviving neuron counts (g). Scale bar 20 μm and magnification 200× in panel a, b and c; Scale bar 10 μm and magnification 400× in panel d, e, and f. (**p < 0.01, ∂∂p < 0.01)

Analysis dendritic morphology of hippocampal CA3 pyramidal neuron

Apical dendritic intersections

Number of apical dendritic intersections studied at 20, 40 and 100 μm was not very much different in RF-EMR exposed group compared to control and sham exposed rats. Statistical tests revealed no difference between the groups in the apical dendritic intersections studied at 20, 40 and 100 μm concentric circles (Fig. 6a). However, the dendritic intersections was observed to be less in RF-EMR exposed group compared to control group at 60 and 80 μm concentric circles studied. ANOVA test revealed that, the decrease in apical dendritic intersections was statistically significant only with control group but not with sham group.
Fig. 6

Apical dendritic intersections (a) and branching points (b), basal dendritic intersections (c) and branching points (d) in CA3 neuron. (*∂p < 0.05, **∂∂p < 0.01, ***∂∂∂p < 0.001)

Apical dendritic branching points

Number of apical dendritic branching points

There was no significant difference in the mean number of apical dendritic branching points at 0–20 and 20–40 μm concentric circles studied. The difference in the mean between groups was not found to be statistically significant. On the other hand, the apical branching points were significantly reduced in RF-EMR exposed between 40 and 60 μm concentric zone compared to control group. Branching points were also found to be reduced at 60-80 and 80-100 μm zones in RF-EMR exposed group compared to control group but not with sham exposed group (Fig. 6b). Total apical branching points were also reduced in RF-EMR exposed group compared to control group but not with Sham exposed group (Fig. 6b).

Basal dendritic intersections

There was a decline in the mean dendritic intersections in RF-EMR exposed group compared to control and sham exposed groups. At 20 μm length the intersections were found to be only slightly less in RF-EMR exposed group compared to controls (Fig. 6c). However, at 40 μm length it was significantly different compared to control but slightly different compared to sham exposed group. The mean number of intersections at 60 and 80 μm lengths was found to be significantly reduced in RF-EMR exposed group compared to control (Fig. 6c). But this difference was found to be only slightly different from the sham exposed group. This pattern was evident even at 100 μm length where the intersections were found to be less in RF-EMR exposed group. However, statistical analysis revealed that, this difference was found to be significant only with control group but not with sham exposed group (Fig. 6c).

Basal dendritic branching points

Number of basal dendritic branching points

The mean number of dendritic branching points was not significantly different in all three groups at concentric zone 0–20 μm. However, at 20–40 μm concentric zone it was significantly reduced in RF-EMR exposed group compared to control but slightly reduced compared to sham exposed group (Fig. 6d). Dendritic branching points were significantly reduced at 40–60 μm concentric zone in RF-EMR exposed group compared other groups (Fig. 6d). Total basal dendritic branching points were also significantly reduced in RF-EMR exposed group compared to other groups (Fig. 6d). ANOVA test revealed a statistically significant difference in the total branching points between groups. The above observations illustrates that, RF-EMR could affect the dendritic arborisation pattern of dorsal hippocampal CA3 pyramidal neurons (Fig. 7c and f). Changes observed in the basal dendritic tree arborisation was striking compared to apical.
Fig. 7

Representative photomicrographs and camera lucida tracings of Golgi-Cox stained hippocampal CA3 neurons from control (a, d) sham exposed (b, e) and RF-EMR exposed (c, f) rats. Scale bar 20 μm, magnification 200× in panel a, b and c. Scale bar 20 μm in panel d, e, and f (Drawn at 400× magnification)

Discussion

To pinpoint ones way around an environment and remembering the events precisely during that process are imperative cognitive capabilities that have been linked to the hippocampal formation and medial temporal lobe (Burgess et al. 2002). Acquisition of new memories is the function of medial temporal lobe and the hippocampus (Scoville and Milner. 1957). In particular, the dorsal hippocampus is crucial for spatial learning in rats but not the ventral. The reason for this is thought to be the presence of higher proportion of place cells and more focused place fields in this brain region compared to the ventral (Jung et al. 1994). It is also suggested that, the difference in connectivity of the dorsal and the ventral hippocampus might be the other reason for this functional difference (Beckstead 1978; Ruth et al. 1982; Witter et al. 1989). The requirement of dorsal hippocampus but not ventral hippocampus for spatial learning has been established by studying spatial learning (using Morris water maze test) in rodents which has only 20 % of the dorsal hippocampus (minislab) (Moser et al. 1995).

In the current study, RF-EMR exposed animals exhibited longer escape latency during the learning sessions compared to control animals. Though their escape latency was elevated during learning trials, over a period of time they had learnt the paradigm as demonstrated by their significantly decreased escape latency on day six compared to the first. The higher escape latency (on day 6) indicates impaired spatial navigation ability in RF-EMR exposed animals compared to controls. In the first retention test, the mean time taken to reach the target quadrant and time spent there were comparable in all three groups. However, number of entries to target quadrant and distance travelled there were slightly decreased in RF-EMR exposed rats. These observations suggest that, RF-EMR exposed animals do learn complex spatial cognition paradigms such as MWM test. Although certain parameters were reduced, RF-EMR exposed rats were able to retrieve this information to some extent whenever required. The crucial questions evolved after these observations are; do they have a clear spatial strategy to pinpoint the exact location of the platform using the extra-maze cues? Are they trying to somehow escape from the maze and in that process due to chance they spent more time in the target quadrant? To answer these questions we conducted a retention test (48 h after the last training session) with a hidden platform kept in the target quadrant as done in acquisition sessions. The results revealed that, RF-EMR exposed rats failed to specifically identify or pin point the platform location. It is also interesting to note that, they did enter the target quadrant by taking almost equal time as control animals but failed to precisely locate the exact position of the hidden platform.

In RF-EMR exposed rats the dorsal hippocampal CA3 region (septal pole) exhibited sparse appearance and degenerating like cells. The surviving neuron count was also found to be reduced in this hippocampal sub-region. Hippocampus is one of the brain regions that is extremely vulnerable to neuronal loss during mild stress (Liu et al. 2010; Ishida et al. 2011) or chronic stress (Sapolsky 2000; Hosseini-Sharifabad et al. 2012). Additional stress due to repeated and chronic RF-EMR exposure would have accelerated this process in the CA3 region. This could be one of the possible reasons for the decreased surviving neuron count in the CA3 region of dorsal hippocampus. These findings in prepubescent rat calls for special attention and concerns because reports suggest that, chronic stress during the peripubertal juvenile period may lead to changes in hippocampal volume in adulthood. Behavioral manifestations such as impaired Morris water maze performance and sustained HPA response to acute stress were also accompanied by this (Isgor et al. 2004). All these differences became apparent only during adulthood and this suggests that stress during adolescence might reduce hippocampal growth (Isgor et al. 2004). Additionally, bilateral lesions of hippocampal septal pole caused spatial learning deficits in rats when tested using Morris water maze (Moser et al. 1995). Recent reports indicate that, information’s about one animal’s motions, routes, within an environment seems to be spatially more extended in CA3 sub-region of hippocampus compared to CA1 (Alvernhe et al. 2008). This indicates the crucial role of septal hippocampal CA3 region in spatial navigation. Since in the current study, cell loss was found with CA3 region, this could be attributed for the altered spatial navigation in RF-EMR exposed rats. A number of reports suggest the possible negative impact of RF-EMR on hippocampal neurons. Odaci et al. (2008) have exposed pregnant rats to RF-EMR (900 MHz; 60 min/day) between the first and last days of gestation and they found an extensive decrease in granule cells count in the dentate gyrus of rat offspring when studied at the age of 4th week. Additionally, GSM 900 MHz RF-EMR exposure with a SAR 0.016 (whole body) and 2 W/kg (locally in the head) for 1 h per day for 28 days induced pyramidal cell loss in the hippocampal CA region of RF-EMR exposed rats (Bas et al. 2009).

Hippocampal neural circuitry shows serial flow of information and is dependent on the integrity of dendritic arborization of CA1 and CA3 neurons. The entorhinal cortex begins this loop by projecting (perforant pathway) to the granule cells of the dentate gyrus. The granule cells send a dense projection of axons (mossy fibers) to the molecular layer of the CA3 subfield where they synapse on the apical dendrites. Axons of CA3 pyramidal neuron bifurcate, one of which enters the alveus to exit the hippocampus in the fibers of the fornix. The collateral axons (schaffer collaterals) project to CA1 pyramidal neurons which in turn project to the subiculum. Finally, pyramidal neurons in the subiculum complete the multisynaptic loop through the hippocampal formation by sending their axons to the entorhinal cortex (Prichard and Alloway 1998). Golgi-Cox staining of hippocampal CA3 pyramidal neurons revealed decreased dendritic complexity suggesting a possible dendritic remodeling in this region under repeated and chronic RF-EMR exposure. Dendritic remodeling in hippocampal CA3 pyramidal neuron has been reported by many in various conditions (Ramkumar et al. 2008; Shankaranarayana Rao et al. 2001; Schloesser et al. 2014). Tsamis et al. (2010) have reported that, both apical and basilar dendritic tree remodeling occurs in Alzheimer’s disease. Reports also support the view that, CA3 neuron atrophy would occurs when chronic stress is applied and this is associated with impaired spatial cognition in rats (Sunanda et al. 2000; Titus et al. 2007). Maskey et al. (2010) have reported that, hippocampal CA3 region of mice showed weak calbindin D28-k (calcium binding protein) immunoreactivity, when exposed chronically to RF-EMR (835 MHz). Calbindin D28-k immunoreactive neurons exhibited decreased dendritic arborization. Decreased pyramidal cells and loss of D28-k immunoreactivity of mossy fibers were also found in RF-EMR exposed group. A probable mechanism for dendritic remodeling in hippocampal CA3 neurons observed in the present study might be RF-EMR induced impaired calcium homeostasis. Impaired calcium homeostasis induced altered dendritic arborization needs special attention. Report suggests that calcium is likely to have key roles in the cellular processes underlying severe dementias, including Alzheimer’s disease (Disterhoft et al. 1994). Altered dendritic arborization seen in the CA3 region might be another possible cause for the impaired cognitive performance of RF-EMR exposed rats. Moreover, an imbalance of different neurotransmitters (glutamate, acetylcholine, dopamine, and serotonin) in the hippocampus has been proposed as the neurobiological basis of behavioural symptoms in some neurodegenerative disorders (Chen et al. 2011). We can only speculate all these at present. Further studies in this regard will be able to unravel the answer. Existence of another possible mechanism, in other words damage of another brain area that leads to impaired spatial learning memory cannot be excluded. Reports suggest that, medial prefrontal cortex plays an important role in spatial memory tasks (Jo et al. 2007). Could repeated and chronic RF-EMR exposure affect the prefrontal cortex function? At present we do not have an answer for this argument because the structural changes in the prefrontal cortex were not studied in the current study. However a report indicates an imbalance in oxidant, antioxidant status in this brain region of rats after chronic RF-EMR (900 MHz) exposure (Narayanan et al. 2014a).

We believe that altered structural integrity in various brain regions (especially hippocampal CA3 region) led to altered behaviour. The mechanism or the cause for the altered structure should also be considered. It could be due to specific effects of RF-EMR or an after effect of stress induced by RF-EMR exposure. Several mechanisms have been proposed to explain the biological effects of RF-EMR on the brain. RF-EMR can have several effects on body systems such as RF-EMR thermal effect, specific effect (non-thermal), or cumulative effect (includes, thermal and non-thermal). RF-EMR thermal effects have been well understood but not the specific effects (non-thermal effects). There are some possible mechanisms suggested to explain the effects of RF-EMR on brain cells which are described below. 1. Increased generation of reactive oxygen species (ROS) (Kesari et al. 2011; Megha et al. 2012), 2. Activation of apoptotic pathway (Zhao et al. 2007), 3. Effects on DNA (Lai and Singh. 1995), 4. Effect on calcium influx/efflux across the membrane (Yu-Hong et al. 2007), 5. Effect on glial cells (Dasdag et al. 2009), 6. Effects on neurotransmitters levels (Khadrawy et al. 2009). Earlier studies have reported that, RF-EMR exposure lead to oxidative damage (Kesari et al. 2013) and monoamine content (Maaroufi et al. 2014) in the hippocampus. It is possible that, the cognitive deficit we have found with RF-EMR exposed group might be due to combined effects of all that happened in various brain regions.

Our observations from the current study provide certain indications for biological effects of mobile phone radiation on prepubescent rats. Growing body of research indicates that, RF-EMR exposure perturb oxidant, antioxidant defence system in the brain (Kesari et al. 2013; Megha et al. 2012). This therefore does not favor appropriate functioning of nerve cells. Whenever the physiological limit is attained, either the cell functions abnormally or dies. We term it as structural change or morphological change. Often what is happening in cellular level is expressed in the behaviour of an organism. However, it is highly difficult to categorically join up biochemical change, morphological change and then behaviour. In the current study we have looked into one of the possibilities, such as ‘hippocampal fragility’ and altered spatial cognition. There might be several other reasons or mechanisms involved in the altered spatial cognition found with rats and these have to be further evaluated. Another crucial question which has to be answered is that, whether RF-EMR induced effects are different in different individuals? This is because there are mechanisms in the body to prevent or resist the insult from external stressors. Currently we do not know this as there are no concrete experimental evidences that pin point or demonstrate this innate preventive/restrain mechanisms present in neurons under RF-EMR exposure. Further study in this regard will reveal much clearer picture of body’s (especially brain) innate mechanisms which would withstand the potential threat caused by RF-EMR.

Conclusion

Chronic and repeated RF-EMR exposure from a mobile phone altered spatial cognition to an extent in rats as demonstrated by their deficits in progressive learning, consolidation and retrieval of spatial information in a spatial cognition testing paradigm. It also induced dendritic remodeling and decreased viable cell number in CA3 region of the hippocampus. Structural changes found in the hippocampus of RF-EMR exposed rats could be one of the possible reasons for altered cognition.

Notes

Acknowledgments

The excellent technical expertise received from Dr. Vasavi Rakesh Gorantla, Assistant Professor and Mr. Raghu Jetty, Senior Grade Lecturer, Department of Anatomy, Melaka Manipal Medical College (Manipal Campus), Manipal University, in brain histology procedures is gratefully acknowledged. Authors also thank Dr. Binu V. S., Associate Professor, Department of Statistics, Manipal University, for his valuable suggestions in statistical analysis. We wish to thank Dr. K. N. Maruthy, Professor, Department of Physiology, Narayana Medical College, Nellore, for designing and fabricating the auto dialer unit for the study. The authors would like to thank Indian Council of Medical Research (ICMR), New Delhi, for partly funding (No. 5/10/FR/21/2011-RHN, IRIS ID: 2011-08800) this research work.

Compliance with ethical standards

Funding

This study was partly funded by Indian Council of Medical Research (ICMR), New Delhi (Grant No. 5/10/FR/21/2011-RHN, IRIS ID: 2011-08800).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sareesh Naduvil Narayanan
    • 1
  • Raju Suresh Kumar
    • 1
    • 5
  • Kalesh M. Karun
    • 2
  • Satheesha B. Nayak
    • 3
  • P. Gopalakrishna Bhat
    • 4
  1. 1.Department of Physiology, Melaka Manipal Medical College (Manipal Campus)Manipal UniversityManipalIndia
  2. 2.Department of StatisticsManipal UniversityManipalIndia
  3. 3.Department of Anatomy, Melaka Manipal Medical College (Manipal Campus)Manipal UniversityManipalIndia
  4. 4.Division of Biotechnology, School of Life SciencesManipal UniversityManipalIndia
  5. 5.College of Science and Health Professions – JeddahKing Saud Bin Abdulaziz University for Health Sciences, National Guard Health AffairsJeddahKingdom of Saudi Arabia

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